Fruit and vegetable discards preserved with sodium metabisulfite as a high-moisture ingredient in total mixed ration for ruminants: effect on in vitro ruminal fermentation and in vivo metabolism.

OBJECTIVE: Our recent series of laboratory- and large-scale experiments confirmed that under aerobic and anaerobic conditions, sodium metabisulfite (SMB) was effective in preserving nutrients and antioxidant capacity of highly perishable fruit and vegetable discards (FVD). Hence, the purpose of this study was to examine how partial inclusion of SMB-treated FVD in total mixed ration (TMR) influences in vitro ruminal fermentation, whole-tract digestibility, nitrogen metabolism, blood metabolites, and voluntary feed intake of sheep. METHODS: The FVD were mixed thoroughly with 6 g SMB/kg wet biomass and kept outdoors under aerobic conditions for 7 days. Four TMRs including four levels of SMB-treated FVD (as-fed basis) at 0%, 10%, 20%, and 30% (equaling to 0%, 1.9%, 3.8%, and 5.7% on dry matter basis, respectively), were prepared as replacement for corn grain. The ruminal fermentation metabolites were studied using an in vitro gas production test. Four mature male Corriedale sheep were assigned at random to the 4 diets for two separate sub-experiments; i) digestibility trial with four 21-d periods, and ii) voluntary feed intake trial with four 28-d periods. RESULTS: Inclusion of SMB-treated FVD in the TMR tended to quadratically increase partitioning factor. No effect was seen on total-tract digestibility of organic matter, ether extract, crude protein, and acid detergent fiber, except for neutral detergent fiber digestibility that tended to linearly increase with increasing SMB-treated FVD in the TMR. The progressive increase of FVD preserved with SMB in the diet had no effect on nitrogen metabolism. Treatment had no effect on serum antioxidant capacity and blood metabolites assayed. Voluntary feed intake was not impaired by inclusion of SMB-treated FVD in the TMR. CONCLUSION: It appears that FVD preserved with SMB can be safely incorporated into TMR as replacement of corn grain without impairment of nutrient metabolism and feed intake.

Heat Stress Effects on Physiological and Blood Parameters, and Behavior in Early Fattening Stage of Beef Steers.

This study was conducted to investigate the effect of heat stress (HS) on physiological, blood, and behavioral parameters, according to the temperature-humidity index (THI), in beef steers. Twelve Korean native beef steers (342.7 ± 13.81 days old, body weight (BW) of 333.0 ± 18.53 kg) were used in this experiment. Beef steers were randomly distributed into three homogenized groups (four beef steers each) for 14 days, namely, threshold (THI = 64-71), mild-moderate (THI = 72-79), and severe (THI = 80-87). Feed and water intake were recorded daily. Physiological parameters, including heart rate and rectal temperature, and behavioral patterns (standing and lying down) were measured weekly. Blood was sampled every week to analyze hormones, heat shock protein (HSP) levels, metabolites, and hematological parameters. All data were analyzed using repeated-measures analysis. Beef steers exposed to severe THI had significantly increased (p < 0.001) water intake, heart rate, and rectal temperature compared to the threshold and mild-moderate THI beef steers. Additionally, increased blood cortisol (p < 0.001), HSP70 (p < 0.001), blood urea nitrogen (BUN) (p = 0.014), and time spent standing (p < 0.001) were observed in beef steers after exposure to severe THI compared to beef steers in the threshold and mild-moderate THI groups. However, dry matter intake, blood glucose, and non-esterified fatty acids were not different among the THI groups. In conclusion, heart rate, rectal temperature, blood cortisol, HSP70, BUN, and time spent standing were closely associated with severe HS conditions in beef steers. These phenomena indicated that beef steers exposed to HS modulated their behavior and blood parameters, as well as their physiological response, to maintain homeostasis.

Developmental arrest of scNT-derived fetuses by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness.

Somatic cell nuclear transfer (scNT)-derived pig placenta tissues of gestational day 30 displayed avascularization and hypovascularization. Most of the cytotrophoblast-like cells of the developing scNT-derived placenta villi were improperly localized or exhibited impaired migration to their targeting loci. Id-2, Met, MMP-9, and MCM-7 were barely detectable in the cytotrophoblast cells of the scNT-derived placenta villi. Active MMP-2 and MMP-9 expression was significantly down-regulated in the scNT-embryo transferred recipient uteri. scNT clones exhibited a hypermethylated pattern within the pig MMP-9 promoter region and the significance of GC box in the regulation of MMP-9 promoter activity. Marked apoptosis was observed in the developing endometrial gland of scNT-embryo transferred recipient uteri. Collectively, our data strongly indicated that early gestational death of scNT clones is caused, at least in part, by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness due to the down-regulation of active MMP-9 expression.

Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development.

Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm.

Essential role of paternal chromatin in the regulation of transcriptional activity during mouse preimplantation development.

Several lines of evidence indicate that the formation of a transcriptionally repressive state during the two-cell stage in the preimplantation mouse embryo is superimposed on the activation of the embryonic genome. However, it is difficult to determine the profile of newly synthesized (nascent) RNA during this phase because large amounts of maternal RNA accumulate in maturing oocytes to support early development. Using 5-bromouridine-5'-triphosphate labeling of RNA, we have verified that nascent RNA synthesis was repressed between the two-cell and four-cell transition in normally fertilized but not in parthenogenetic embryos. Moreover, this repression was contributed by sperm (male) chromatin, which we confirmed by studying androgenetic embryos. The source of factors responsible for repressing nascent RNA production was investigated using different stages of sperm development. Fertilization with immature round spermatids resulted in a lower level of transcriptional activity than with ICSI at the two-cell stage, and this was consistent with further repression at the four-cell stage in the ICSI group. Finally, study on DNA replication and chromatin remodeling was performed using labeled histones H3 and H4 to differentiate between male and female pronuclei. The combination of male and female chromatin appeared to decrease nascent RNA production in the fertilized embryo. This study indicates that paternal chromatin is important in the regulation of transcriptional activity during mouse preimplantation development and that this capacity is acquired during spermiogenesis.

Determination of in situ degradation parameters and feeding level of pineapple (Ananas comosus L.) cannery by-product to Hanwoo steers.

OBJECTIVE: The objectives of this study were to determine the in situ degradation parameters and appropriate feeding level of pineapple cannery by-products (PCB) based on the growth performance and blood parameters of growing Hanwoo (Bos taurus coreanae) steers fed various levels of PCB. METHODS: Two ruminally cannulated Holstein cows were used for in situ disappearance rate measurements. Nylon bags (5×10 cm, 45 µm pore size) filled with 5 g of PCB in triplicate were inserted into the ventral sac of the two cannulated Holsteins cows and incubated for 0, 2, 4, 8, 16, 24, and 48 h. A total of 16 castrated growing Hanwoo steers (12.5±0.5 months old, 302.9±25.7 kg of initial body weight [BW]) were used for the experiment. Animals were stratified by initial BW and then randomly assigned to one of four experimental diets (0%, 1.5%, 3.0%, or 6.0% of PCB, on the dry matter [DM] basis) fed for 91-d, including 30-d of adaptation. RESULTS: Soluble fraction a of DM and crude protein (CP) was 61.9% and 86.0%, fraction b of DM and CP was 32.7% and 11.2%, and indigestible fraction c of DM and CP was 5.4% and 2.8%. The 6.0% PCB feeding group showed lower productivity compared to animals in the other treatments. Increasing the dietary level of PCB did not alter DM intake, but it was numerically lowest in the 6.0% feeding group. The gain to feed ratio was linearly decreased by increasing of PCB. The quadratic broken-line test estimated that 2.5% (DM basis) was the maximum feeding level of PCB in growing Hanwoo steers (y = 0.103 - 0.001×[1.245-x]2, R2 = 0.18). CONCLUSION: Diets containing up to 2.5% PCB can be fed to growing Hanwoo steers without adverse effects on growth performance.

Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos.

Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5'-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.

Long-term anaerobic conservation of fruit and vegetable discards without or with moisture adjustment after aerobic preservation with sodium metabisulfite.

The high moisture and soluble carbohydrates contents of fruit and vegetable discards accelerate putrefaction; thus, the immediate use of preservatives is necessary to prevent their decay and allow further use. Two series of experiments were performed to evaluate the conservation stability of fruit and vegetable discards through ensiling. Fruit and vegetable discards were not treated or treated with 6 g sodium metabisulfite/kg fruit and vegetable discards and aerobically challenged for 7 days. In Experiment 1, sodium metabisulfite-treated fruit and vegetable discards were ensiled alone as high-moisture biomass. Silage fermentation was not effective in preventing the deterioration and mold control, which resulted in the rapid depletion of sugars and dry matter loss in control silage (without sodium metabisulfite). Conversely, the low number of undesirable microorganisms resulted in the negligible loss of nutrients in the sodium metabisulfite-treated silage. In Experiment 2, sodium metabisulfite-treated fruit and vegetable discards were co-ensiled with dry by-product feeds at varying proportions to provide sodium metabisulfite loads of 1.6, 2.4, 3.2, and 4 g/kg biomass. Based on microbiological, nutrient composition, and organoleptic evaluations, the sodium metabisulfite load of 3.2 g/kg biomass provided the most desirable conservation parameters. This study provides a clean route to the safe utilization of the discards for a prolonged period, with negligible dry matter and nutrient loss.

Dietary effects of lutein-fortified chlorella on milk components of Holstein cows.

This study was conducted to investigate the dietary effect of conventional or lutein-fortified chlorella on milk production and lutein incorporation in milk. Fifteen Holstein cows in mid-lactation were used in a 3 × 3 Latin square design each with a 21-day period. Cows were top-dressed daily with 30 g of conventional or lutein-fortified chlorella for 3 weeks. Cows without chlorella served as the control. The feed intake and milk yield were not affected by dietary treatments. The concentrations of milk protein and solids non-fat in groups fed diets containing both conventional and lutein-fortified chlorella were significantly higher than those of the control group (P < 0.001). There was no significant difference in content of milk fat among groups. The levels of plasma glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, interferon-gamma and interleukin-2 were not influenced by the dietary treatments. Lutein content in milk was significantly increased in groups fed lutein-fortified chlorella as compared with those of conventional chlorella and control, respectively (P < 0.01). These results imply that conventional and lutein-fortified chlorella has positive effects on milk components and the use of lutein-fortified chlorella in a dairy diet is effective in the production of milk enriched with lutein.

Induction of castration by immunization of male dogs with recombinant gonadotropin-releasing hormone (GnRH)-canine distemper virus (CDV) T helper cell epitope p35.

Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets.

Effects of various anesthetic protocols on 18F-flurodeoxyglucose uptake into the brains and hearts of normal miniature pigs (Sus scrofa domestica).

This study used positron emission tomography-computed tomography (PET-CT) to evaluate the effects of 4 anesthetic protocols on 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) accumulation in the brains and hearts of miniature pigs (Sus scrofa domestica). The 18F-FDG standard uptake value was quantified by dividing the brain into 6 regions: cerebellum, brainstem, and frontal, parietal, temporal, and occipital lobes. Five (2 female and 3 male) clinically normal miniature pigs were premedicated with medetomidine (200 µg/kg IM) after which the following 4 anesthetic protocols were administered by using a crossover design: 1) propofol (4 mg/kg IV)-isoflurane inhalation; 2) propofol (4 mg/kg IV); 3) ketamine (5 mg/kg IV); 4) tiletamine-zolazepam (4.4 mg/kg IM). Compared with levels after other protocols, brain accumulation of 18F-FDG increased during propofol anesthesia but decreased with tiletamine-zolazepam. Relative to that due to other protocols, heart accumulation of 18F-FDG increased with propofol-isoflurane anesthesia but decreased with tiletamine-zolazepam. Comparing glucose accumulation in the brain and heart of miniature pigs by using PET-CT, we found that glucose accumulation varied according to the anesthetic protocol and between the 2 organs. These results can be used to evaluate how different anesthetic agents affect glucose metabolism in brain and heart of miniature pigs. Furthermore, these data should be considered when selecting an anesthetic agent for miniature pigs that will undergo PET-CT imaging with 18F-FDG.

Discrimination of animal species using polymorphisms of the nuclear gene zinc finger protein 238.

We screened 3750 single exonic genes listed in the intronless genes in the eukaryotes (SEGE) database and performed bioinformatic analyses to identify candidate genes for new species-specific markers. A set of PCR primers for the conserved regions of ZNF238 was developed and used to amplify the 823 bp DNA fragment. We compared nucleotide variations of the PCR products among 20 species plus two subspecies of animals, which led to the identification of interspecies nucleotide variations. To establish a simple method for the analysis of species-specific DNA polymorphisms using ZNF238, we developed a PCR-RFLP method using HhaI and HpyCH4IV restriction enzymes for 13 species. For the remaining species, the direct sequencing of PCR products provided additional SNPs, enabling precise species classification. As a result, we report here that a new nuclear DNA marker, ZNF238, can be used to increase the accuracy of species identification among euteleostomi (bony vertebrates).

Depigmentation of skin and hair color in the somatic cell cloned pig.

Previously, we have successfully produced nine cloned piglets using Duroc donor cells. Among these clones, one showed distinct depigmentation of the skin and hair color during puberty. In this study, we selected a clone with depigmentation to investigate the etiology of the anomaly in somatic cell nuclear transfer. We hypothesized that genes related to Waardenburg syndrome (Mitf, Pax-3, Sox-10, Slug, and Kit) are closely associated with the depigmentation of pig, which was derived from somatic cell nuclear transfer (scNT). Total RNA was extracted from the ear tissue of affected and unaffected scNT-derived pigs, and the transcripts encoding Mitf, Pax-3, Sox-10, and Slug, together with the Kit gene, were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed. The cDNA sequences from the scNT pig that showed progressive depigmentation did not reveal a mutation in these genes. Although we did not find any mutations in these genes, expression of the genes implicated in Waardenburg syndrome was severely down-regulated in the affected scNT pig when compared with unaffected scNT pigs. This down-regulation of gene expression may result in a previously undescribed phenotype that shows melanocyte instability, leading to progressive loss of pigmentation.